Project Abstract: Successful in vitro hematopoietic stem cell (HSC) expansion would be therapeutically invaluable in transplant- based therapies, but it has proven to be a formidable challenge, owing in part to our limited understanding of how HSCs expand in vivo. The complicated origins and migratory development of fetal HSCs have made this difficult task even more daunting. The fetal liver is an important developmental site of physiological HSC expansion, however the molecular mechanisms that drive the proliferation of these stem cells in the fetal liver are not understood. In recent studies, we have investigated the role of Notch signaling during fetal development using a knock-in mouse model lacking the transcriptional activation domain of Notch1 (DTAD). We have discovered a requirement for Notch1 in the viability of HSCs during fetal liver expansion and a novel role for Notch signaling in fetal HSCs following transplantation in a recipient. In this Research Project Grant, we will build on these findings by carrying out in depth investigation of Notch signaling in fetal HSCs and in the fetal microenvironment. In Aim1, we will elucidate the molecular mechanism of the Notch signaling apparatus in the fetal liver HSCs. We predict that a combination of specific protein interactions and unique transcriptional targets direct the cell fate decisions of fetal HSCs and that their interactions and targets differ from adult bone marrow HSCs. By relying on the Notch1DTAD mutant, and generating a conditional Notch1DTAD mouse model, we will identify the tissue specific Notch transcriptional machinery essential for survival in the fetal liver and for the reconstitutive potential of HSCs. In Aim 2, we will investigate the fetal liver microenvironment that provides the framework for tissue specific expansion of fetal HSCs. We will examine various population of fetal liver cells and test them for the surface expression of Notch ligands. Endothelial cells (FL ECs) express high levels of Notch ligands Jagged1 and Dll4, thus we will determine their role in the activation of Notch signaling in the fetal liver. We will use in vivo transgenic animal models and an in vitro co-culture system to test the expansion of HSCs through the use of Notch-ligand presenting FL ECs and the integration of the Notch co-culture system with other established signaling pathways. The knowledge gained from these aims will enhance our understanding of how signaling pathways drive stem cell maintenance, expansion as well function in a transplantation setting. Uncovering the signaling cues by which HSCs expand without exhausting their self- renewal capacity is of immense therapeutic importance in overcoming both blood disorders related to aging and disease.